The present invention relates to a method for measuring the concentration of a polynucleotide such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) in a sample using agglutination of an agent, and to a kit and apparatus for use in such a method.
Recent innovation in the field of genetic engineering has been remarkable. In particular, the development of a polymerase chain reaction (PCR) method has enabled the mass replication of target DNA. The PCR method is based on the principle that DNA polymerase does not function without a primer. The PCR method is used to amplify DNA in large amounts by repeating the following cycle: (1) DNA in a sample is heat denaturated resulting in single stranded DNA; (2) a primer is bonded to the DNA under reduced temperature; and (3) the DNA is formed by thermo-stable DNA polymerase under this condition. Consequently, target DNA can be prepared in large amounts by using a specific primer prepared by chemical synthesis or otherwise. In this method, the replication of target DNA is typically monitored by electrophoresis, a dot blotting method, a PCR-SSCP (Single Strand Conformation Polymorphism) method, a PCR-fluorescence method or the like.
In gene analysis, electrophoresis is a generally used method for detecting the mobility of polynucleotide in a gel in the presence of an electric field. The dot blotting method is used to judge whether or not the amount of analyte has increased or has decreased by the following steps: (1) the polynucleotide extracted from a sample is gradually diluted and the equivalent amount is spotted to a nitrocellulose filter or a nylon film; (2) DNA, RNA and the like labeled by the radioisotope (32P) are hybridized with the polynucleotide; and (3) the polynucleotide is exposed to an X-ray film and is analyzed, or the spot portion of film is cut off after hybridization and measured by a scintillation counter. The PCR-SSCP method is a method for detecting the position of a band by autoradiography by the following steps: (1) sample DNA is amplified by the PCR method using the primer labeled by the radioisotope (32P); (2) the labeled DNA fragment obtained is heat denaturated resulting in single stranded DNA; and (3) single stranded DNA is separated by electrophoresis using a neutral polyacrylamide gel. The PCR-fluorescence method comprises a first step of obtaining the initial amount of the target nucleic acid by following the change in the fluorescence intensity after PCR has been carried out in the presence of an intercalating fluorescence material (Progress in Medical, Vol. 173, No. 12, Jun. 17, 1995, Pages 959-963).
However, these analytical methods suffer the drawback that special apparatus is needed, the manipulation is complicated and takes a long time. Moreover, the type of polynucleotide which may be analyzed is limited with respect to the chosen analytical method.
More specifically, electrophoresis is a complex and lengthy technique in which it is necessary to prepare a gel as a carrier and to pre-select a gel (size) for which PCR was carried out with regard to the size of DNA. Typically, a sample takes about 75 minutes to electrophorese and it is therefore not a rapid technique.
In the dot blotting method and the PCR-SSCP method, there is normally a safety factor because of the use of a radioisotope. In the method in which no radioisotope is used, the DNA probe must be labeled with a fluorescent substance or a luminescent material. This makes the method complicated and it takes a long time for the manipulation of film transfer. Furthermore, in the PCR-fluorescence method a fluorophotometer is needed and it is difficult to detect single stranded DNA. The conventional analytical methods have in common the problem that the analysis can not be carried out in the presence of materials other than polynucleotide. Therefore, a sample for which PCR has been carried out (a PCR product) should be purified prior to analysis. This makes the overall manipulation lengthy.
It is an object of the present invention to seek to solve these problems and others in the known techniques for measuring polynucleotide concentration in a sample.
Thus viewed from one aspect the present invention provides a method for measuring the presence of polynucleotide in a sample comprising the steps of: (1) preparing a sample, an agglutinative agent and an agglutination promoter capable of binding to polynucleotide; (2) mixing said sample, said agent and said promoter; and (3) measuring the degree of agglutination of the agent. Preferably the method is used to determine the concentration of said polynucleotide.
The present invention is based on the principle that agglutination of the agent is not promoted by the promoter when the promoter is bound to a polynucleotide. Therefore, by measuring the degree of agglutination, it is possible to confirm the presence of polynucleotide in the sample and in a preferred embodiment to measure its concentration. The measurement of the degree of agglutination may be readily carried out by measuring the absorbance with a spectrophotometer or simply by visual observation. In this invention, mixing the sample with the agent and the promoter is a simple step. Moreover, the method of the invention as a whole may be carried out in a short period of time, since the agglutination of the agent typically occurs in e.g. 1-2 seconds and the measurement of the degree of agglutination may be equally carried out in a short time. Furthermore, the concentration of polynucleotide in a sample may be measured by the method of the invention, even if the materials other than polynucleotide are included in the sample e.g. PCR product that has not been purified.
In the present invention, the expression xe2x80x9can agglutination promoter capable of binding to polynucleotidexe2x80x9d refers to material which has the characteristics of bonding to polynucleotide and of promoting the agglutination of the agglutinative agent and which can not promote the agglutination of the agent when bound to polynucleotide. The term xe2x80x9can agglutinative agentxe2x80x9d in the present invention refers to material that agglutinates rapidly in the presence of the promoter.
The promoter and the agent may be added to the sample at the same time, or consecutively e.g. the promoter may be added before the agent.
The polynucleotide under investigation may be, for example, single stranded DNA, double stranded DNA, RNA, a complex of RNA and DNA or PNA (Peptide nucleic acid), etc. The method of the present invention is effective on a sample which has been subjected to the PCR method (PCR product) or on a DNA sample. As mentioned above, the PCR method is used for amplifying target DNA. If target DNA is not present in the sample, trace amounts of DNA would be found in the PCR product. Therefore, by measuring the degree of agglutination in accordance with the method of the present invention, it may be determined simply and rapidly whether or not target DNA has been amplified. Further, the amount of amplified DNA can be measured provided a calibration curve relating the degree of agglutination and the amount of DNA has been prepared beforehand.
In addition, the method of the present invention may usefully be applied to samples in which the amplification of DNA is carried out by the Strand displacement amplification method (SDA method) or by the Ligase chain reaction (LCR method) and in which the amplification of RNA is carried out using the Qxcex2 replicase (Qxcex2 method). The SDA method includes the method described in Nucleic Acids Research, Vol.20. No.7 1691-1696. The LCR method is that method in which thermo-stable DNA ligase which is not denaturated by heat (even at 94xc2x0 C.) is used. This method is based on the principle that for normal DNA samples having no mismatches, the two kinds of oligonucleotide used are bonded by the DNA ligase, resulting in them functioning as a substrate in the next reaction cycle and DNA amplification. Where the DNA sample has mismatches, the reaction stops, since the two kinds of oligonucleotide are not able to bond.
A RNA replicase which has high specificity to substrate RNA is used in the Qxcex2 method. Firstly, the plasmid vector connected to MDV-1-RNA (RNA changing to DNA) is placed downstream of the promoter of T7 RNA polymerase and the DNA fragment to be amplified is inserted into the MDV-1 (the fragment of about 20-800bp can be inserted into it). After it is cut by using a restriction enzyme, the DNA fragment is charged to RNA by using T7 RNA polymerase. The MDV-1-RNA in which the DNA fragment is inserted can be amplified by repeating the replicative cycle with Qxcex2 replicase.
In addition, the method of the present invention may usefully be applied to samples in which the amplification of DNA or RNA is carried out by a 3SR method, a NASBA method, a CPR method, a SIR method or the like. The NASBA method is an RNA replication method and the other methods are DNA replication methods.
For measuring the concentration of double stranded DNA (including a sample treated by a DNA replication method) in accordance with the invention, the agent may be 4xe2x80x2,6-diamidino-2-phenylindole (DAPI) which is shown in formula 1 below, ethidium bromide (EtBr), thiazole orange, bisbenzimide (Hoechst 33258, product of Hoechst AG) which is shown in formula 2 below and acridine orange. In addition, SYBR Green I (Molecular Probes Co., Ltd.) may be included. It is preferable that the chosen agent is DAPI, since it bonds to double stranded DNA to form a complex but barely bonds to other material such as RNA. Therefore, double stranded DNA can be selectively detected by DAPI. 
For measuring the concentration of RNA (including RNA amplifying sample) in accordance with the invention, the promoter may be e.g. EtBr, thiazole orange, bisbenzimide (Hoechst 33258, product of Hoechst AG) which is shown in formula 2 and acridine orange. Additionally, SYBR Green II (Molecular Probes Co., Ltd.) may be included. Among the materials mentioned above, SYBR Green II bonds to RNA more selectively to form a complex.
For measuring the concentration of single stranded DNA in accordance with the invention SYBR Green II may be used as the promoter.
For measuring the concentration of a complex of RNA and DNA an agent which bonds to DNA and/or RNA may be used as the promoter
In the present invention, the agent may be, for example, a silver colloid, a gold colloid, a copper colloid, or a latex which may be simply manufactured and easily treated. The latex may be, for example, a coloured latex, a carboxylated latex or an aminated latex. A commercial product may be used as the silver colloid and the like in the method of the invention. The silver colloid and the like may be prepared in a conventional manner.
In the measuring step, it is preferably to measure the degree of agglutination by straightforward visual observation. If it is desired to measure the concentration of polynucleotide, it is preferable to determine the degree of agglutination by measuring the absorbance with a spectrophotometer thereby enabling accurate measurement.
Viewed from a further aspect the present invention provides a kit comprising a reagent R1 comprising an agglutination promoter capable of bonding to polynucleotide and a reagent R2 comprising an agglutinative agent. The measurement of the concentration of polynucleotide may be carried out more rapidly and easily by using this kit.
Viewed from a yet further aspect the present invention provides an apparatus comprising: (1) means for introducing a reagent R1 comprising an agglutination promoter capable of bonding to polynucleotide into a sample; (2) means for introducing a reagent R2 comprising an agglutinative agent into the mixture produced in step (1); and (3) a system for measuring the absorbance of radiation by the agglutination. This apparatus enables the measurement of the concentration of polynucleotide to be carried out automatically.